Methods of inhibiting helicobacter pylori

ABSTRACT

This invention relates to methods of screening molecules capable of inhibiting the survival of  Helicobacter pylori  in vivo by specifically inhibiting the activity of UreI, to the molecules identified by these methods, and to the use of these molecules to treat or prevent  H. pylori  infection.

This application is a continuation of international application numberPCT/EP99/04490, filed Jun. 29, 1999, and is a continuation of U.S.application Ser. No. 09/107,383, filed Jun. 30, 1998 now U.S. Pat.6,190,667 the content of which is incorporated herein by reference.

This invention relates to methods of screening molecules capable ofinhibiting the survival of Helicobacter, particularly Helicobacterpylori, in vivo by specifically, inhibiting the activity of UreI, to themolecules identified by these methods, and to the use of these moleculesto treat or prevent Helicobacter infection.

BACKGROUND OF INVENTION

Helicobacter priori is a microaerophilic Gram-negative bacterium, whichcolonizes the gastric mucosa of humans (10). H. pylori is associatedwith gastritis and peptic ulcer disease and has been shown to increasethe risk of gastric cancers. Urease is a major virulence factor of H.pylori. It is involved in neutralizing the acidic microenvironment ofthe bacterium and also plays a role in H. pylori metabolism (11, 26).

The urease region of the H. pylori genome is composed of two geneclusters common to all strains (9 and FIG. 1), one comprising the ureABgenes encoding the structural urease subunits and the other containingthe ureEFGH genes encoding the accessory proteins required for nickelincorporation into the urease active site. The UreI gene liesimmediately upstream from this latter gene cluster and is transcribed inthe same direction (FIG. 1). The ureA, ureB, ureE, ureF, ureG, ureH, andureI genes and gene products have been described and claimed in U.S.Pat. No. 5.695,931 and allowed patent application Ser. No. 08/472,285.both of which are specifically incorporated herein by reference.

The distances separating UreI from ureE (one base pair, bp) and ureEfrom ureF (11 bp) suggest that ureI-ureE-ureF constitute an operon.Cotranscription of ureI and ureE has been demonstrated by northern blotanalysis (1). An H. pylori N6 mutant with a ureI gene disrupted by aMiniTn3-Km transposon was previously described by Ferrero et al. (1994)(13). This strain (N6-ureI::TnKm-8) presented a urease negativephenotype, so it was concluded that ureI was an accessory gene requiredfor full urease activity.

The sequences of UreI from H. pylori and the AmiS proteins, encoded bythe aliphatic amidase operons of Pseudomonas aeruginosa and Rhodococcussp. R312, are similar (5, 27). Aliphatic amidases catalyze theintracellular hydrolysis of short-chain aliphatic amides to produce thecorresponding organic acid and ammonia. It has been shown that H. pylorialso has such an aliphatic amidase, which hydrolyzes acetamide andpropionamide in vitro (23).

In view of the sequence similarity between UreI and AmiS together withthe very similar structures of the urease and amidase substrates (urea:NH₂—CO—NH₂ and acetamide: CH₃—CO—NH₂) and the production of ammonia byboth enzymes, a better understanding of the function of the H. pyloriUreI protein is required. This understanding will open new opportunitiesfor the prevention and treatment of H. pylori infections.

SUMMARY OF THE INVENTION

This invention provides methods for identifying molecules capable ofinhibiting the growth and/or survival of Helicobacter species,particularly, H. pylori, in vivo. In particular, the methods of thisinvention involve screening molecules that specifically inhibit UreIprotein function.

The invention encompasses the molecules identified by the methods ofthis invention and the use of the molecules by the methods of thisinvention to treat or prevent Helicobacter, and particularly H. pylori,infection in humans and animals.

Another aspect of this invention is a method of preventing or treatingHelicobacter species infection by administration to a human or animal inneed of such treatment a molecule capable of inhibiting the growthand/or survival of Helicobacter species in vivo. One such moleculeaccording to the invention is characterized by a high affinity for UreI,which allows it (i) to be transported inside the Helicobacter cell, or(ii) to inhibit transport properties of UreI, or (iii) to inhibit UreIfunction by inhibiting UreI interaction with urease or otherHelicobacter proteins. By inhibiting UreI, such molecule renders thebacteria more sensitive to acidity.

Yet another aspect of this invention is the production of immunogenicUreI antigens and their use as vaccines to prevent Helicobacter speciesinfection and/or colonization of the stomach or the gut. Antibodies tothese UreI antigens are also encompassed within the scope of thisinvention.

This invention further relates to recombinant strains of H. pyloricomprising a modified ureI gene, such that the products of the modifiedgene contribute to the attenuation of the bacteria's ability to survivein vivo and thus, its pathogenic effects.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the urease gene cluster of H. pylori parental strains N6and SS1 and of the derived mutants deficient in UreI, strains N6-823,N6-834, and SS1-834. The genes are indicated by boxes Kith an arrowshowing the direction of their transcription. The distances between theure genes are given in base pairs, bp. The site hybridizing to theprimers used to confirm correct allelic exchange in strains N6-823,N6-834, and SS1-834 is shown. Blank boxes represent the cassettescontaining the genes conferring resistance to Cm (cat) or to Km(aphA-3). The urease activity of these strains is given on theright-hand side of the figure. Urease activity was measured as therelease of ammonia on crude extracts of bacteria grown 48 hours on bloodagar plates as described previously (9). One unit corresponds to theamount of enzyme required to hydrolyze 1 μmol of urea min⁻¹ mg⁻¹ totalprotein. The data are means±standard deviation calculated from 3 to 5determinations.

FIG. 2A depicts a restriction map of p1LL823, pILL824, p1LL833 andp1LL834. Small boxes mark the vector of each plasmid, and large boxescorrespond to genes. On indicates the position of the ColE1 origin ofreplication. Sp^(R) and AP^(R) are the genes conferring resistance tospectinomycin and ampicillin, respectively. Cassettes inserted into ureland conferring resistance to chloramphenicol (cat) or kanamycin (aphA-3)are also shown. The sequence of the DNA region (SEQ ID NO: 17)comprising the urel stop codon and the ureE start codon, including theBcll site where adaptor H19 (SEQ ID NO: 18) was inserted, is given.Insertion of H19 into the Bcll site of p1LL824 produced p1LL825, theresulting urel-ureE intergenic region is also shown. The stop codon ofurel and the start codon of ureE are boxed and the ribosome binding site(RBS) is underlined. Brackets indicate the position of restriction sitesremoved by ligation.

FIG. 2B depicts a restriction map of two H. pylori/E. coli shuttleplasmids: pILL845 and pILL850. Small boxes mark the vector of eachplasmid, and large boxes correspond to genes. Ori indicates the positionof the E. coli ColE1 origin of replication and repA the gene coding forthe RepA protein necessary for autonomous replication of the pHe12 in H.pylori. Cm^(P) a marks the gene conferring resistance tochloramphenicol. The ureI promoter is represented by a “P” with an arrowindicating the direction of the transcription. The other symbols are asin FIG. 1.

FIG. 3 shows the alignment of the amino acid sequence of Urel from H.pylori with those of similar proteins and prediction of thetwo-dimensional structure of members of the Urel/AmiS protein family.Residues identical at one position in, at least, four sequences areboxed and dashes indicate gaps inserted to optimize alignment. Theorganisms from which the sequences originated and the degree of identitywith the H. pylon Urel protein are: UreI-Hp, Helicocobacter pylori (195residues, accession No. M84338) (SEQ ID NO: 10); Urel-Hf, Helicobacterfelis (74% identity over 196 residues, accession No. A41012) (SEQ ID NO:11); Urel-Lacto, Lactobacillus fermentum (55% identity over the 46residues-long partial sequence, accession No. D10605) (SEQ ID NO: 12);Urel-Strepto, Streptococcus salivarius (54% identity over the 129residues-long partial sequence, accession No. U35248) (SEQ ID NO: 13);AmiS-Myco, Mycobacterium smegmatis (39% identity over 172 residues,accession No. X57175) (SEQ ID NO: 14); AmiS-Rhod, Rhodococcus sp. R312(37% identity over 172 residues, accession No. Z46523) (SEQ ID NO: 15),and AmiS-Pseudo, Pseudomonas aeruginosa (37% identity over 171 residues,accession No. X77161) (SEQ ID NO: 16). Predicted transmembrane -helicesare shown as shaded boxes. The regions separating these boxes arehydrophilic loops labeled “IN” when predicted to be intracellular and“OUT” when predicted to be extracellular.

FIG. 4 depicts the kinetics of ammonium release by the N6 parentalstrain (panel A) and the UreI-deficient strain N6-834 (panel B).Bacteria (2×10⁸/ml) were harvested and washed (as described inSkouloubris et al. (30)) resuspended in 10 ml of phosphate saline buffer(PBS) at pH 7.5 to 2.2 in the presence of 10 mM urea. After 0, 3, 5 and30 minutes, 0.5 ml were withdrawn and centrifuged to eliminate bacteria.The supernatant was kept on ice until ammonium concentration wasmeasured using the assay commercialized by Sigma (kit reference #171).

Table 2 shows the results obtained with the in vitro viability tests andthe pH measurements.

Table 3 gives the values of ammonium production by strain N6 and N6-834presented on the graphs of FIG. 4.

DETAILED DESCRIPTION

The urease cluster of Helicobacter species is unique among the manyurease operons of Gram-negative bacteria that have been sequenced (20)in that it has an extra gene, UreI. The function of UreI has thereforebeen the subject of much speculation. It has mostly been attributed thefunction of an accessory protein required for nickel incorporation atthe urease active site or a nickel transporter. A H. pylori straincarrying a deletion of ureI replaced by a non-polar cassette (Kanamycinresistance cassette) has been constructed and was named N6-834 (30). Thestrain has been deposited at C.N.C.M. (Collection Nationale de Culturede Microorganismes, 25 rue du Docteur Roux, 75724 Paris Cédex 15,France) on Jun. 28, 1999. This is the first time that a non-polarcassette (19) has been shown to be functional in H. pylori. Theseresults provide a valuable tool for genetic analysis of complex H.pylori operons, such as Cag. a multigenic pathogenicity island.

Studies with this strain demonstrated that UreI is not required for fullactivity of H. pylori urease as measured after in vitro growth atneutral pH. This result argues against UreI being involved in nickeltransport since such a protein, NixA (3) already identified in H.pylori, is necessary for full urease activity. Comparing ureasesexpressed from a UreI-deficient strain and the corresponding parentalstrain show that (i) they present the same activity optimum pH (pH 8);(ii) the urease structural subunits, UreA-B, are produced in equalamounts; and (iii) the urease cellular location is identical.

It is demonstrated here that (i) UreI is essential for colonization ofmice by H. pylori; (ii) UreI is important for survival of H. pylori atacidic pH; and (iii) UreI is necessary for urease “activation” at lowpH.

H. pylori during the colonization process of the stomach has to dealwith important pH variations and especially has to adapt rapidly toextremely acidic pH (as acidic as pH 1.4). We have shown that UreI isrequired for H. pylori adaptation to acidity, consistently with theabsence of colonization of the mouse stomach. As an essential proteinfor the H. pylori resistance to acidity, UreI certainly plays a key rolein the infection, establishment, and persistence of H. pylori. UreI hasa sequence similar to those of the AmiS proteins, proposed to beinvolved in the transport of short-chain amides (27), moleculesstructurally similar to urea. The UreI/AmiS proteins have thecharacteristics of integral membrane proteins, probably of thecytoplasmic membrane.

Different roles for UreI can be proposed. For instance, UreI might beinvolved in transport (import or export) of urea or short chain amidesspecifically active at low pH. However, an essential role for UreI as anamide transporter is less likely because a SS1 mutant, deficient inaliphatic amidase, colonizes as efficiently as the parental strain inmouse colonization experiments. In addition, amidase activity is notsignificantly modified by the deletion of ureI in the N6-834 mutantstrain (C.N.C.M. filed on Jun. 28,1999). Import or export of urea couldbe consistent with the existence of a urea cycle, which is one of thecharacteristics of H. pylori (28).

Alternatively, UreI might be involved in an active ammonium exportsystem. Finally, UreI might be involved in a mechanism of couplingurease activity to the periplasmic pH, allowing urease to become moreactive when extracellular pH is acidic.

Our results are compatible with the first hypothesis of UreI being anurea transporter active at acidic pH values and the third hypothesis ofUreI being a kind of sensor protein between the periplasmic pH andurease activity. We think that these two hypothesis are not exclusive.Whatever the role of UreI, as a membrane protein essential for thesurvival of H. pylori in vivo, it now provides a powerful target for anew eradication therapy and for vaccines against H. pylori.

Molecules capable of inhibiting the growth and/or survival ofHelicobacter in vivo may be identified by contacting a parentalHelicobacter strain with said molecule in a biological sample; testingand comparing, in the presence or absence of urea, the sensitivity tothe extracellular pH of the parental strain to a strain deficient inUreI and to a UreI deficient strain complemented with ureI selectingsaid molecules displaying a differential effect on the parental orcomplemented strain as compared to the UreI deficient strain; andcollecting said active molecule.

A molecule active specifically on UreI will be the one rendering H.pylori sensitive to acidic pH (pH 2.2) in the presence of urea withoutaffecting the strain behavior at neural pH. Sensitivity to acidity inthe presence of urea can be tested on whole H. pylori cells following aprotocol described in the examples and adapted from Clyne et al. (8). Weare now trying to transpose this test in E. coli whole cells carryingthe complete urease gene cluster on a plasmid (ureAB-ureIEFGH).Screening for a molecule rendering this recombinant E. coli moresensitive to acidity in the presence of urea will be performed asdescribed for H. pylori in the examples. To distinguish betweeninhibitory molecules acting on UreI and those acting on urease, themedium pH after whole cell incubation at pH 7 in the presence of ureawill be measured. Interesting molecules are those affecting response toacidity without inhibiting the alkalization of the medium observed afterincubation at neutral pH.

These methods may be used to identify molecules that inhibit anyHelicobacter species carrying a UreI-homolog. This includes the gastricHelicobacter species: Helicobacter pylori, Helicobacter felis,Helicobacter mustelae, Helicobacter muridarum, and also Helicobacterheiimannii, Helicobacter canis, Helicobacter bilis, Helicobacterhepaticus, and Helicobacter troguntum.

The molecules identified by the methods of this invention will becapable of inhibiting UreI activity by (i) inhibiting transport of ureaor short chain amides, (ii) inhibiting ammonium export, or (iii)inhibiting urease “activation” at low pH. The molecules according topoint (i) and (ii) should be able to diffuse throughout the outermembrane and should be active even at low concentration. Suitablecandidate molecules are structural analogs of urea or short chainamides, ammonium derivatives or urease inhibitors. For example,molecules derived from AHA (acetohydroxamic acid), hydroxyurea, hippuricacid, flurofamide, hydroxylamine, methylurea, thiourea (29), ormethylammonium. The molecules according to point (iii) should inhibitthe contact between UreI (probably inserted in the cytoplasmic membrane)and periplasmic, membrane, or cytoplasmic H. pylori proteins, which arenecessary for urease “activation” at low pH. These proteins could be thestructural subunits of urease itself, the accessory proteins, or otherproteins. Molecules obtained according to this invention should not beurease competitive inhibitors, should not be toxic or mutagenic in vivoand could potentialize the action of antibiotics or bactericidalmolecules. Validation of the action of such molecules could be performedin vivo in the mouse animal model with the pair of isogenic strains SS1and SS1-834 as described in the examples.

One example of a molecule according to this invention is a monoclonal orpolyclonal antibody specific for UreI. Preferably, the antibody iscapable of specifically inhibiting UreI activity.

The molecules of this invention may be administered in combination witha pharmaceutically acceptable carrier to a patient suffering from aHelicobacter infection. Alternatively, immunogenic compositionscomprising one or more molecules according to this invention may beadministered in a vaccine composition to prevent infection byHelicobacter species.

Immunogenic compositions according to this invention may also compriseall or part of the UreI protein. Preferably, the UreI fragments compriseat least 10 consecutive amino acids of the native UreI sequence and morepreferably, the fragments comprise at least 18, 20, or 25 consecutiveamino acids of the native UreI sequence. Other suitable UreI fragmentsmay contain at least 40 or at least 100 consecutive amino acids of thenative UreI sequence. Suitable fragments of Helicobacter pylori include,for example, fragments selected from the group consisting of amino acidresidues 22 to 31, 49 to 74, 94 to 104, and 123 to 142 of H. pylori(GenBank accession No. M84338)

Reference will now be made to the following Examples. The Examples arepurely exemplary of the invention and are not to be construed aslimiting of the invention.

EXAMPLES

Construction of defined mutations of the H. pylori ureI gene

H. pylori strains with defined mutations in ureI were generated byallelic exchange to determine whether the UreI protein was necessary forproduction of active urease. For this purpose, two plasmids (pILL823 andpILL834) with cassettes carrying antibiotic resistance genes inserted inureI were constructed in E. coli.

In one plasmid. pILL823 (FIG. 2A), the urel gene was inactivated byinsertion of a promoterless cat gene. conferring resistance tochloramphenicol (Cm). A 780 bp blunt-ended BamHI restriction fragmentcontaining the “cat cartridge” from pCM4 (Pharmacia, Sweden) wasintroduced into a unique HpaI site, between codons 21 and 22 of ureI, inpILL753 (9). In the resulting plasmid, pILL823 (FIG. 2A), cat is in thesame orientation as ureI and is expressed under the control of the ureIpromoter.

The second plasmid, pILL834, carried a UreI gene in which all but thefirst 21 codons were deleted and replaced with a non-polar cassettecomposed of the aphA-3 kanamycin (Km) resistance gene (25), which hasbeen deleted from its own promoter and terminator regions (19). InShigella flexneri (19) and other organisms (such as Yersiniaenterocolitica, 2) this cassette has been shown not to affect thetranscription of the genes downstream within an operon as long as thesedistal genes have intact translation signals. There is only one basepair separating ureI from ureE (FIG. 1) and ureE does not have an RBS(ribosome binding site) of its own, so the expression of ureI and ureEis transcriptionally and translationally coupled. Therefore, a ureIdeletion was accompanied by the addition of an RBS immediately upstreamfrom ureE. Three intermediates, pILL824, pILL825 and pILL833 (FIG. 2A),were constructed in order to produce the final plasmid, pILL834 (FIG.2A). A 1.8 Kb HpaI-HindIII restriction fragment from pILL753 (9) wasinserted between the EcoRV and HindIII sites of pB3922, to give pILL824.insertion of the H19 adaptor (carrying an RBS and ATG in frame withureE, Table I) into a BclI site overlapping the two first codons of ureEin pILL824 produced pILL825 (FIG. 2A). The BamHI fragment of pILL825 wasthen replaced by a 1.3 Kb blunt-ended PvuII-BamHI fragment from pILL753.This resulted in the reconstitution of a complete ureI gene, and thisplasmid was called pILL833. Finally, pILL834 was obtained by replacementof the HpaI-BglII fragment of pILL833 (thereby deleting all but thefirst 21 codons of ureI) with an 850 bp blunt-ended EcoRI-BamHI fragmentof pUC18K2 containing the non-polar Km cassette (19).

TABLE 1 Name and nucleotide sequence of oligonucleotidesOligodeoxynucleotide Primer sequence (5′ to 3′) H17TTTGACTTACTGGGGATCAAGCCTG (SEQ ID NO:1) H19*GATCATTTATTCCTCCAGATCTGGAGGAATAAAT (SEQ ID NO:2) H28GAAGATCTCTAGGACTTGTATTGTTATAT (SEQ ID NO:3) H34 TATCAACGGTGGTATATCCAGTG(SEQ ID NO:4) H35 GCAGTTATTGGTGCCCTTAAACG (SEQ ID NO:5) H50CCGGTGATATTCTCATTTTAGCC (SEQ ID NO:6)  8A GCGAGTATGTAGGTTCAGTA (SEQ IDNO:7)  9B GTGATACTTGAGCAATATCTTCAGC (SEQ ID NO:8) 12BCAAATCCACATAATCCACGCTGAAATC (SEQ ID NO:9) *H19 was used as adaptor andthe others were used as primers for PCR amplification.

Introduction of ureI mutations into H. pylori

H. pylori ureI mutants were produced by allelic exchange followingelectroporation with a concentrated preparation of pILL823 and pILL834as previously described by Skouloubris et al. (23) from H. pylori strainN6 (12) and from the mouse-adapted H. pylori strain SS1 (Sydney Strain17). Bacteria with chromosomal allelic exchange with pILL883 wereselected on Cm (4 μml) and those with chromosomal allelic exchange withpILL834 on Km (20 μg/ml). It was determined that the desired allelicexchange had taken place in strains N6-823, N6-834. and SS1-834 (FIG. 1)by performing PCR with the appropriate oligonucleotides (Table 1). ThePCR products obtained with genomic DNA of these strains were as expected(i) for strain N6-823: 140 bp with primers H28-H34, 220 bp with H35-9B,and 1.2 Kb with H28-9B, and (ii) for strains N6-834 and SS1-834, 150 bpwith primers H28-H50, 180 bp with H17-12B, and 1 Kb with H28-12B.

The growth rate of strain N6-834 carrying a non-polar deletion of ureIwas compared to that of the parental strain N6. No difference in thecolony size was observed on blood agar medium plates. Identical doublingtimes and stationary phase OD were measured for both strains grown inBHI (Oxoid) liquid medium containing 0.2% ∃-cyclodextrin (Sigma). Thus,UreI is not essential for H. pylori growth in vitro.

Urease activity, of H. pylori ureI mutants

The urease activity of strains N6-823, N6-834. and SS1-834 was measuredin vitro as described previously by Cussac et al. (9) and compared tothe activity of the parental strains, N6 and SS 1 (FIG. 1). 7Ureaseactivity was almost completely abolished in strain N6-823 (0.3±0.1units). Strains N6-834 and SS l -834, with non-polar ureI mutations hadwild-type levels of activity (N6-834 and SS1-834: 12-2 units; parentalstrains, N6: 10±1 and SS1: 12±0.4 units).

The pH optimum of urease produced either from the N6 parental strain orfrom the UreI deficient strain N6-834 was measured and compared. Forboth strains, urease has a pH optimum of 8 which is consistent with thepublished data.

These results strongly suggest that the urease-negative phenotype of theN6-ureI::TnKm-8 (13) and the very weak urease activity of N6-823 strainswere due to a polar effect of the inserted cassettes on the expressionof the downstream genes ureE and ureF (FIG. 1). This hypothesis wastested by measuring urease activity of strain N6-823 complemented intrans with an E. coli/H. pylori shuttle plasmid expressing the ureE-Fgenes. This plasmid, pILL845 (FIG. 2B), was obtained by insertion of a2.8 Kb ClaI-BamHI fragment of pILL834 (comprising the 3′-end of ureB,the non-polar deletion of ureI and intact ureE and ureF genes) into thecorresponding sites of the shuttle vector pHe12 constructed by Heuermannand Haas (15). Strain N6-823 was electroporated with a DNA preparationof pILL845 as described by Skouloubris et al. (23), and transformantswere selected on kanamycin (20 μg/ml) and chloramphenicol (4 μg/ml). instrain N6-823 harboring pILL845, wild type urease activity was recoveredconfirming that the very low urease activity of strain N6-823 was due toa polar effect on the expression of the accessory genes ureE-F. InKlebsiella aerogenes, the absence of UreE has little effect on ureaseactivity (4). In contrast, UreF, as part of the accessory proteincomplex (UreDFG), is absolutely required for the production of activeurease (21). Thus, by analogy, it is likely that the phenotype of the H.pylori polar ureI mutants was due to the absence of ureF expression.

The urease structural subunits, UreA and UreB, produced by strain N6 orstrain N6-834 were compared with the Western blot technique using amixture of antisera directed against each urease subunit. It wasobserved that the amount of each subunit produced by the two strains isidentical. The possibility that urease cellular localization could beaffected in the absence of UreI was examined after cellularfractionation (separating the soluble from the membrane associatedproteins and from the supernatant) of strains N6 and N6-834. Theseexperiments revealed no difference between the urease cellularlocalization in the wild type strain or in the UreI-deficient mutant.These results demonstrate that, at neutral pH, UreI is neitherimplicated in the stabilization of the urease structural subunits nor ina targeting process of urease to a specific cellular compartment.

Colonization test for the H. pylori SS1-834 mutant in the mouse animalmodel

The mouse model for infection by the H. pylori SS1 strain (SydneyStrain, 17), validated by Chevalier et al. (7) and Ferrero et al. (14),was used to test the function of UreI in vivo. Mice were infected withthe non-polar ureI mutant, SS 1-834, and with the parental strain, SS1,(which had gone through an equivalent number of in vitro subcultures) asa positive control. This experiment was repeated three times andproduced identical results (30). Two independently constructed SS-834mutants were used. The first mutant strain had gone through 30 in vitrosubcultures, the second only 20. Under the same experimental conditions,strain SS1 can undergo more than 80 in vitro subcultures without losingits colonization capacity.

In each experiment, aliquots (100 μl) containing 10⁶ H. pylori strainSS1 or SS1-834 bacteria prepared in peptone broth were administeredorogastrically to 10 mice each (six to eight-weeks old Swissspecific-pathogen-free mice) as described by Ferrero et al. (I14). Micewere killed four weeks after inoculation. The presence of H. pylori wastested with a direct urease test on biopsies performed on half thestomach (14). The remaining gastric tissues were used for quantitativeculture of H. pylori as described by Ferrero et al. (14). In eachexperiment, the stomachs of the ten SS1-infected mice all testedpositive for urease. The bacterial load was between 5×10⁴ and 5×10⁵colony forming units (CFU) per g of stomach. None of the stomachs of themice infected with strain SS1-834 tested positive for urease and no H.pylori cells were cultured from them. Thus, the UreI protein isessential for the H. pylori in vivo survival and/or colonization of themouse stomach.

UreI is essential for H. pylori resistance to acidity

Survival to acidic conditions in the presence or absence of 10 mM ureawas tested with strains N6 and N6-834. The experimental proceduresdetailed in Skouloubris et al. (30) were based on those described inClyne et al. (8). Exponentially grown bacteria were harvested, washed inPBS (phosphate buffer saline), and approximately 2×10⁸ CFU/ml wereresuspended in PBS of pH 2.2 or pH 7 in the presence or the absence of10 mM urea and incubated at 37EC. After one hour incubation (i)quantitative cultures of the H. pylori strains were performed toevaluate bacterial survival, and (ii) the bacteria were centrifuged andthe pH of the medium was measured. The results obtained are presented inTable 2. In the absence of urea, both strains N6 and N6-834 presentedidentical phenotype, i.e., they were killed at pH 2.2, and survived atpH 7 without modifying the final pH of the medium (Table 2). Afterincubation at pH 7 in the presence of urea, both strains were killedbecause the final pH rose to pH 9. At pH 2.2 in the presence of urea,the parental strain survived well since it was able to raise the pH toneutrality. In contrast, a completely different phenotype was obtainedwith the UreI-deficient strain N6-834 which was unable to raise the pHand whose viability was seriously affected (Table 2).

Complementation of the UreI-deficient strain N6834 with plasmid pILL850

Direct implication of the UreI protein in the H. pylori capacity toresist to acidity has been confirmed by trans-complementation withplasmid pILL850 (FIG. 2B restriction map and details of construction).This plasmid [CNCM I-2245 filed on Jun. 28,1999] is derived from the H.pylori/E. coli shuttle vector pHe12 (15). Plasmid pILL850 carries theureI gene under the control of its own promoter and was constructed asfollows: a 1.2 kb BclI restriction fragment of plasmid pILL753 (9) wasintroduced between the BamHI and BclI restriction sites of pHe12 (FIG.2B). Strains N6 and N6-834 were transformed by this plasmid and thephenotype of the complemented strains in the acidity sensitivity testexperiments described above was examined. As shown in Table 2, thephenotype of strain N6-834 complemented by pILL850 is identical to thatof the parental strain N6. Interestingly, the urease activity of thecomplemented strains (measured on sonicated extracts as described inSkouloubris et al. (30)) has been found to be significantly higher ascompared to that of the corresponding strains without pILL850. For thepurpose of the deposit at the CNCM pILL850 is placed into an E. colistrain, MC1061 (Wertman K. F. et al, 1986, Gene 49: 253-262).

Measurements of ammonium production

The amount of ammonium produced in the extracellular medium of H. pyloriwhole cells was measured by an enzymatic assay commercialized by Sigmafollowing the supplier's instructions. These experiments were performedafter incubation of the cells in PBS at different pH values and afterdifferent incubation times. Such experiments gave an accurate evaluationof ammonium production and excretion in different strains as well as ameasure of the kinetics of this reaction. A control experiment showedthat ammonium production was very low (10-20 μM) in the absence of urea.

FIG. 4 depicts the kinetics (0, 3, 5, and 30 min. incubation time) ofextracellular ammonium released by the N6 parental strain (panel A) andthe UreI-deficient strain N6-834 (panel B) incubated in PBS at pH 2.2,pH 5, or pH 7 in the presence of 10 mM urea. The results obtainedindicate that (i) ammonium is largely produced and rapidly released inthe extracellular medium; and (ii) in the N6 wild type strain (FIG. 4,panel A and Table 3) ammonium production is significantly enhanced whenthe extracellular pH is acidic. This effect is already visible at pH 5and is even stronger at pH 2.2. This last observation is consistent withthe results of Scott et al. (31) who suggested urease activation at lowpH. In our experiments, the rapidity of the response to acidity arguesagainst urease activation depending on transcriptional regulation or onde novo protein synthesis.

Ammonium production was then measured in the UreI-deficient strainN6-834 (FIG. 4, panel B and Table 3). At neutral pH, kinetics ofammonium production were similar to those of the wild type strain. Incontrast, at pH 5 ammonium production was reduced and delayed ascompared to the wild type strain. A dramatic effect of the absence ofUreI was observed at pH 2.2, where the amount of ammonium was very low,which is consistent with our results showing that UreI is necessary foradaptation to acidity.

Our results demonstrate that UreI is essential for the resistance of H.pylori to acidity. In the absence of UreI, urease, although present inhuge amounts, is not able to protect the bacteria against the aggressionof acidity. This is consistent with the essential role of UreI in vivo.During its passage in the acidic stomach lumen, the viability of theUreI-deficient strain is affected. As a consequence, the bacterial loadbecomes too low to permit colonization. The different roles proposed forUreI are presented in the “detailed description” section.

Alignment of the UreI and AmiS protein sequences and two dimensionalstructure prediction

A systematic search for UreI homologs in the protein data banks wascarried out. It was determined that H. pylori is not the only ureolyticbacterium with a ureI gene. Two phylogenetically related Gram-positiveorganisms, Streptococcus salivarius, a dental plaque bacterium (6), andLactobacillus fermentum, a lactic acid bacterium (16), carry genes forUreI-homologs (FIG. 3) located immediately upstream from the ureasestructural genes. The ureI gene has been detected in variousHelicobacter species; the H. felis ureI gene has been entirely sequenced(FIG. 3 and allowed U.S. patent application Ser. No. 08/467,822, theentire contents of which are incorporated herein by reference). PCRexperiments have suggested that there is a ureI gene in H. heilmannii(24) and in H. mustelae.

Sequence similarities between the UreI protein of H. pylori and the AmiSproteins expressed by the aliphatic amidase operons from P. aeruginosa(27) and Rhodococcus sp. R312 (5) have been reported. In Mycobacteriumsmegmatis, there is an additional AmiS-homolog encoded by a gene, ORFP3, located immediately upstream from an amidase gene (18).

Alignment of these UreI/AmiS proteins [using the Clustal W(1.60)program] defined strongly conserved stretches of amino acids (FIG. 3).AU but one of these conserved blocks are in highly hydrophobic segments.These regions, each 17 to 22 residues long, are probably folded intotransmembrane ∀-helices (FIG. 3). Six transmembrane regions werepredicted for the proteins from H. pylori, H. felis, and P. aeruginosaand seven for those from Rhodococcus sp. R312 and M. smegmatis (highlyreliable predictions, performed with pHD, a profile fed neural networksystem as described by Rost et al. (22)). The orientation of theUreI/AmiS proteins in the membrane was deduced from the charges of theintercalated hydrophilic regions, which are short in these proteins(FIG. 3). The first five such regions are poorly conserved and ofvarious length. The last interhelical segment common to these proteinsis significantly more conserved than the others. This region predictedto be intracellular may be the active site of UreI or a site ofmultimerization or interaction with an intracellular partner. Theseresults strongly suggest that the members of the UreI/AmiS family, foundin both Gram-positive and -negative bacteria, are integral membraneproteins. These proteins have no signal sequence and should therefore beinserted into the cytoplasmic membrane in Gram-negative bacteria.

Two peptides, selected from the UreI sequence, were synthesized andinjected into two rabbits to obtain serum containing polyclonalantibodies directed against UreI. One peptide corresponds to the firstpredicted intracellular loop of UreI (from residue nB 15 to 31, see FIG.3) and the second one to the second predicted extracellular loop of UreI(from residue nB 118 to 134, see FIG. 3. These sera are presently beingtested and if proven to recognize the UreI protein will allow us toprecisely define the localization of this protein and to verify thepredicted UreI two-dimensional structure presented in FIG. 3.

The references cited herein are specifically incorporated by referencein their entirety.

TABLE 2 Effect of the presence of urea at pH 7, 5 or 2.2 on (i) theviability of different H. pylori strains and (ii) the extracellular pH(indicated as final pH). The experimental procedures are described inreference 30 and in the examples. Strain N6 is the parental strain andstrain N6-834 the Urel-deficient mutant. Plasmid pILL850 is derived froma E. coli/ H. pylori shuttle vector, it carries the urel gene andcomplements the urel mutation of strain N6-834. urea strains initial pHfinal pH 10 mM H. pylori CFU/ml N6 2.2 2.26 − 0 N6 2.2 6.6 +   8 × 10⁷N6 7 6.98 −   2 × 10⁸ N6 7 8.88 + 0 N6-834 2.2 2.2 − 0 N6-834 2.2 2.37 +  7 × 10⁵ N6-834 7 7.1 − 3.5 × 10⁷ N6-834 7 9.05 + 0 N6-834 + pILL8502.2 2.3 − 0 N6-834 + pILL850 2.2 6.9 + 1.3 × 10⁸ N6-834 + pILL850 7 7.1− 1.7 × 10⁸ N6-834 + pILL850 7 9 + 0

TABLE 3 medium [NH4] Strain pH minutes mM N6 7,0 0 3.5 N6 7,0 3 4.4 N67,0 5 3.1 N6 7,0 30 5.6 N6 5,0 0 12.8 N6 5,0 3 9.3 N6 5,0 5 11.8 N6 5,030 16,0 N6 2,2 0 6.7 N6 2,2 3 9,0 N6 2,2 5 11,0 N6 2,2 30 20,0 N6-8347,0 0 2,.7 N6-834 7,0 3 2.8 N6-834 7,0 5 3.8 N6-834 7,0 30 5.8 N6-8345,0 0 1.4 N6-834 5,0 3 1.7 N6-834 5,0 5 2.9 N6-834 5,0 30 4.6 N6-834 2,20 0.9 N6-834 2,2 3 0.6 N6-834 2,2 5 0.7 N6-834 2,2 30 1.3

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5. Chebrou, H., F. Bigey, A. Arnaud, and P. Galzy. 1996. Amidemetabolism: a putative ABC transporter in Rhodococcus sp. R312. Gene.182:215-218.

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13. Ferrero, R. L., V. Cussac, P. Courcoux, and A. Labigne. 1994.Construction of isogenic mutants of Helicobacter pylori deficient inurease activity. pp 179-182. In Basic and Clinical Aspects of H. pyloriinfection. Springer-Verlag Berlin Heidelberg.

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27. Wilson, S. A., R. J. Williams, L. H. Pearl, and R. E. Drew,. 1995.Identification of two new genes in the Pseudomonas aeruginosa amidaseoperon, encoding an ATPase (AmiB) and a putative integral membraneprotein (AmiS). J. Biol. Chem. 270:18818-18824.

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                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 18 <210> SEQ ID NO 1 <211> LENGTH: 25<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 1 tttgacttac tggggatcaa gcctg          #                   #               25 <210> SEQ ID NO 2<211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Adaptor       sequence <400> SEQUENCE: 2gatcatttat tcctccagat ctggaggaat aaat        #                  #        34 <210> SEQ ID NO 3 <211> LENGTH: 29 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 3 gaagatctct aggacttgta ttgttatat         #                   #            29 <210> SEQ ID NO 4 <211> LENGTH: 23<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 4 tatcaacggt ggtatatcca gtg           #                   #                23 <210> SEQ ID NO 5<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 5 gcagttattg gtgcccttaa acg           #                   #                23 <210> SEQ ID NO 6<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 6 ccggtgatat tctcatttta gcc           #                   #                23 <210> SEQ ID NO 7<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 7 gcgagtatgt aggttcagta            #                   #                   # 20 <210> SEQ ID NO 8<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 8gtgatacttg agcaatatct tcagc           #                  #               25 <210> SEQ ID NO 9 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 9 caaatccaca taatccacgc tgaaatc          #                   #             27 <210> SEQ ID NO 10<211> LENGTH: 195 <212> TYPE: PRT <213> ORGANISM: Helicobacter pylori<400> SEQUENCE: 10 Met Leu Gly Leu Val Leu Leu Tyr Val Gly Il#e Val Leu Ile Ser Asn   1               5  #                 10 #                 15 Gly Ile Cys Gly Leu Thr Lys Val Asp Pro Ly#s Ser Thr Ala Val Met              20      #             25     #             30 Asn Phe Phe Val Gly Gly Leu Ser Ile Ile Cy#s Asn Val Val Val Ile          35          #         40         #         45 Thr Tyr Ser Ala Leu Asn Pro Thr Ala Pro Va#l Glu Gly Ala Glu Asp      50              #     55             #     60 Ile Ala Gln Val Ser His His Leu Thr Asn Ph#e Tyr Gly Pro Ala Thr  65                  # 70                 # 75                  # 80 Gly Leu Leu Phe Gly Phe Thr Tyr Leu Tyr Al#a Ala Ile Asn His Thr                  85  #                 90 #                 95 Phe Gly Leu Asp Trp Arg Pro Tyr Ser Trp Ty#r Ser Leu Phe Val Ala             100       #           105      #           110 Ile Asn Thr Ile Pro Ala Ala Ile Leu Ser Hi#s Tyr Ser Asp Met Leu         115           #       120          #       125 Asp Asp His Lys Val Leu Gly Ile Thr Glu Gl#y Asp Trp Trp Ala Ile     130               #   135              #   140 Ile Trp Leu Ala Trp Gly Val Leu Trp Leu Th#r Ala Phe Ile Glu Asn 145                 1 #50                 1#55                 1 #60 Ile Leu Lys Ile Pro Leu Gly Lys Phe Thr Pr#o Trp Leu Ala Ile Ile                 165   #               170  #               175 Glu Gly Ile Leu Thr Ala Trp Ile Pro Ala Tr#p Leu Leu Phe Ile Gln             180       #           185      #           190 His Trp Val         195 <210> SEQ ID NO 11<211> LENGTH: 196 <212> TYPE: PRT <213> ORGANISM: Helicobacter felis<400> SEQUENCE: 11 Met Leu Gly Leu Val Leu Leu Tyr Val Ala Va#l Val Leu Ile Ser Asn   1               5  #                 10 #                 15 Gly Val Ser Gly Leu Ala Asn Val Asp Ala Ly#s Ser Lys Ala Ile Met              20      #             25     #             30 Asn Tyr Phe Val Gly Gly Asp Ser Pro Leu Cy#s Val Met Trp Ser Leu          35          #         40         #         45 Ser Ser Tyr Ser Thr Phe His Pro Thr Pro Pr#o Ala Thr Gly Pro Glu      50              #     55             #     60 Asp Val Ala Gln Val Ser Gln His Leu Ile As#n Phe Tyr Gly Pro Ala  65                  # 70                 # 75                  # 80 Thr Gly Leu Leu Phe Gly Phe Thr Tyr Leu Ty#r Ala Ala Ile Asn Asn                  85  #                 90 #                 95 Thr Phe Asn Leu Asp Trp Lys Pro Tyr Gly Tr#p Tyr Cys Leu Phe Val             100       #           105      #           110 Thr Ile Asn Thr Ile Pro Ala Ala Ile Leu Se#r His Tyr Ser Asp Ala         115           #       120          #       125 Leu Asp Asp His Arg Leu Leu Gly Ile Thr Gl#u Gly Asp Trp Trp Ala     130               #   135              #   140 Phe Ile Trp Leu Ala Trp Gly Val Leu Trp Le#u Thr Gly Trp Ile Glu 145                 1 #50                 1#55                 1 #60 Cys Ala Leu Gly Lys Ser Leu Gly Lys Phe Va#l Pro Trp Leu Ala Ile                 165   #               170  #               175 Val Glu Gly Val Ile Thr Ala Trp Ile Pro Al#a Trp Leu Leu Phe Ile             180       #           185      #           190 Gln His Trp Ser         195 <210> SEQ ID NO 12<211> LENGTH: 46 <212> TYPE: PRT <213> ORGANISM: Lactobacillus fermentum<400> SEQUENCE: 12 Ile Leu Trp Leu Thr Gly Phe Leu Thr Asn As#n Leu Lys Met Asn Leu   1               5  #                 10 #                 15 Gly Lys Phe Pro Gly Tyr Leu Gly Ile Ile Gl#u Gly Ile Cys Thr Ala              20      #             25     #             30 Trp Ile Pro Gly Phe Leu Met Leu Leu Asn Ty#r Trp Pro Asn          35          #         40          #         45<210> SEQ ID NO 13 <211> LENGTH: 129 <212> TYPE: PRT<213> ORGANISM: Streptococcus salivarius <400> SEQUENCE: 13Ile Leu Asn Ile Ile Val Ile Ala Tyr Gly Al #a Cys Thr Gly Gln Gly  1               5  #                 10  #                 15Ala Glu Trp Phe Tyr Gly Ser Ala Thr Gly Le #u Leu Phe Ala Phe Thr             20      #             25      #             30Tyr Leu Tyr Ser Ala Ile Asn Thr Ile Phe As #p Phe Asp Gln Arg Leu         35          #         40          #         45Tyr Gly Trp Phe Ser Leu Phe Val Ala Ile As #n Thr Leu Pro Ala Gly     50              #     55              #     60Ile Leu Cys Leu Thr Ser Gly Tyr Gly Gly As #n Ala Trp Tyr Gly Ile 65                  # 70                  # 75                  # 80Ile Trp Phe Leu Trp Gly Ile Leu Trp Leu Th #r Ala Phe Ile Glu Ile                 85  #                 90  #                 95Asn Leu Lys Lys Asn Leu Gly Lys Phe Val Pr #o Tyr Leu Ala Ile Phe            100       #           105       #           110Glu Gly Ile Val Thr Ala Trp Ile Pro Gly Le #u Leu Met Leu Trp Gly        115           #       120           #       125 Lys<210> SEQ ID NO 14 <211> LENGTH: 213 <212> TYPE: PRT<213> ORGANISM: Mycobacterium smegmatis <400> SEQUENCE: 14Met Gly Gly Val Gly Leu Phe Tyr Val Gly Al #a Val Leu Ile Ile Asp  1               5  #                 10  #                 15Gly Leu Met Leu Leu Gly Arg Ile Ser Pro Ar #g Gly Ala Thr Pro Leu             20      #             25      #             30Asn Phe Phe Val Gly Gly Leu Gln Val Val Th #r Pro Thr Val Leu Ile         35          #         40          #         45Leu Gln Ser Gly Gly Asp Ala Ala Val Ile Ph #e Ala Ala Ser Gly Leu     50              #     55              #     60Tyr Leu Phe Gly Phe Thr Tyr Leu Trp Val Al #a Ile Asn Asn Val Thr 65                  # 70                  # 75                  # 80Asp Trp Asp Gly Glu Gly Leu Gly Trp Phe Se #r Leu Phe Val Ala Ile                 85  #                 90  #                 95Ala Ala Leu Gly Tyr Ser Trp His Ala Phe Th #r Ala Glu Ala Asp Pro            100       #           105       #           110Ala Phe Gly Val Ile Trp Leu Leu Trp Ala Va #l Leu Trp Phe Met Leu        115           #       120           #       125Phe Leu Leu Leu Gly Leu Gly His Asp Ala Le #u Gly Pro Ala Val Gly    130               #   135               #   140Phe Val Ala Val Ala Glu Gly Val Ile Thr Al #a Ala Val Pro Ala Phe145                 1 #50                 1 #55                 1 #60Leu Ile Val Ser Gly Asn Trp Glu Thr Gly Pr #o Leu Pro Ala Ala Val                165   #               170   #               175Ile Ala Val Ile Gly Phe Ala Ala Val Val Le #u Ala Tyr Pro Ile Gly            180       #           185       #           190Arg Arg Leu Ala Ala Pro Ser Val Thr Asn Pr #o Pro Pro Ala Ala Leu        195           #       200           #       205Ala Ala Thr Thr Arg     210 <210> SEQ ID NO 15 <211> LENGTH: 206<212> TYPE: PRT <213> ORGANISM: Rhodococcus sp. <400> SEQUENCE: 15Met Gly Ser Val Gly Leu Leu Tyr Val Gly Al #a Val Leu Phe Val Asn  1               5  #                 10  #                 15Gly Leu Met Leu Leu Gly Thr Val Pro Val Ar #g Ser Ala Ser Val Leu             20      #             25      #             30Asn Leu Phe Val Gly Ala Leu Gln Cys Val Va #l Pro Thr Val Met Leu         35          #         40          #         45Ile Gln Ala Gln Gly Asp Ser Ser Ala Val Le #u Ala Ala Ser Gly Leu     50              #     55              #     60Tyr Leu Phe Gly Phe Thr Tyr Leu Tyr Val Gl #y Ile Ser Asn Leu Ala 65                  # 70                  # 75                  # 80Gly Phe Glu Pro Glu Gly Ile Gly Trp Phe Se #r Leu Phe Val Ala Cys                 85  #                 90  #                 95Ala Ala Leu Val Tyr Ser Phe Leu Ser Phe Th #r Val Ser Asn Asp Pro            100       #           105       #           110Val Phe Gly Val Ile Trp Leu Ala Trp Ala Al #a Leu Trp Thr Leu Phe        115           #       120           #       125Phe Leu Val Leu Gly Leu Gly Arg Glu Asn Le #u Ser Arg Phe Thr Gly    130               #   135               #   140Trp Ala Ala Ile Leu Leu Ser Gln Pro Thr Cy #s Thr Val Pro Ala Phe145                 1 #50                 1 #55                 1 #60Leu Ile Leu Thr Gly Asn Phe His Thr Thr Pr #o Ala Val Ala Ala Gly                165   #               170   #               175Trp Ala Gly Ala Leu Leu Val Leu Leu Gly Le #u Ala Lys Ile Leu Ala            180       #           185       #           190Ala Pro Lys Ala Ala Val Pro Gln Pro Arg Pr #o Val Phe Asn        195           #       200           #       205<210> SEQ ID NO 16 <211> LENGTH: 171 <212> TYPE: PRT<213> ORGANISM: Pseudomonas aeruginosa <400> SEQUENCE: 16Met Leu Gly Leu Val Leu Leu Tyr Val Gly Al #a Val Leu Phe Leu Asn  1               5  #                 10  #                 15Ala Val Trp Leu Leu Gly Lys Ile Ser Gly Ar #g Glu Val Ala Val Ile             20      #             25      #             30Asn Phe Leu Val Gly Val Leu Ser Ala Cys Va #l Ala Phe Tyr Leu Ile         35          #         40          #         45Phe Ser Ala Ala Ala Gly Gln Gly Ser Leu Ly #s Ala Gly Ala Leu Thr     50              #     55              #     60Leu Leu Phe Ala Phe Thr Tyr Leu Trp Val Al #a Ala Asn Gln Phe Leu 65                  # 70                  # 75                  # 80Glu Val Asp Gly Lys Gly Leu Gly Trp Phe Cy #s Leu Phe Val Ser Leu                 85  #                 90  #                 95Thr Ala Cys Thr Val Ala Ile Glu Ser Phe Al #a Gly Ala Ser Gly Pro            100       #           105       #           110Phe Gly Leu Trp Asn Ala Val Asn Trp Thr Va #l Trp Ala Leu Leu Trp        115           #       120           #       125Phe Cys Phe Phe Leu Leu Leu Gly Leu Ser Ar #g Gly Ile Gln Lys Pro    130               #   135               #   140Val Ala Tyr Leu Thr Leu Ala Ser Ala Ile Ph #e Thr Ala Trp Leu Pro145                 1 #50                 1 #55                 1 #60Gly Leu Leu Leu Leu Gly Gln Val Leu Lys Al #a                 165  #               170 <210> SEQ ID NO 17 <211> LENGTH: 19 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 17 tgggtgtgag atgatcata              #                  #                   # 19 <210> SEQ ID NO 18 <211> LENGTH: 53<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Adaptor      sequence <400> SEQUENCE: 18tgggtgtgag atgatcattt attcctccag atctggagga ataaatgatc at#a            53

We claim:
 1. A plasmid pILL 850, deposited at the Collection Nationalede Culture de Microorganismes, on Jun. 28, 1999 under Accession NumberI-2245.